The present disclosure relates to sample preparation, centrifugal separation, and microfluidic processing of fluids.
Pathogen detection in whole blood samples using molecular techniques requires a sample treatment process which yields a suspension of target nucleic acids which is sufficiently free of PCR inhibitors, interferents and non-target nucleic acids. The sample treatment process is closely tied to the amplification and detection techniques utilized and as such are vital to sensitive and specific detection of target microbes. For instance, the number of target microbial cells in whole blood, on the order of 101 CFU/mL, is vastly outnumbered by blood cells, on the order of 1010/mL. Blood cells are therefore sources of large amount of background DNA, PCR inhibitors, RNase, and fluorescence quenchers. Moreover, dead microbes and nucleic acid from such microbes may also be present in the sample from previously treated infections. This imposes strict functionality requirements on the nucleic acid based pathogen detection platforms.
Existing methods of performing sample preparation on whole blood samples typically consist of the following steps: (i) the blood sample is subjected to some means of lysing the blood cells and microbial cells, either selectively or non-selectively with respect to the target microbes; (ii) removal or inactivation of inhibitors and interferents to PCR and detection; and (iii) removal of non-target nucleic acid or enhanced amplification and detection strategies for increasing specificity with respect to target microbes and live versus dead microbes.
These steps are typically performed either separately or in combination and with varying levels of efficacy in accordance with the tolerance characteristics of downstream processes. Most existing pathogen detection platforms rely on extraction and purification of the target nucleic acids prior to amplification and detection using PCR or RT-PCR, and are poorly-suited for automation in applications involving low pathogen concentrations.